New role of bone morphogenetic protein 7 in brown adipogenesis and energy expenditure

Abstract

Adipose tissue is central to the regulation of energy balance. Two functionally different types of fat are present in mammals: white adipose tissue, the primary site of triglyceride storage, and brown adipose tissue, which is specialized in energy expenditure and can counteract obesity. Factors that specify the developmental fate and function of white and brown adipose tissue remain poorly understood. Here we demonstrate that whereas some members of the family of bone morphogenetic proteins (BMPs) support white adipocyte differentiation, BMP7 singularly promotes differentiation of brown preadipocytes even in the absence of the normally required hormonal induction cocktail. BMP7 activates a full program of brown adipogenesis including induction of early regulators of brown fat fate PRDM16 (PR-domain-containing 16; ref. 4) and PGC-1alpha (peroxisome proliferator-activated receptor-gamma (PPARgamma) coactivator-1alpha; ref. 5), increased expression of the brown-fat-defining marker uncoupling protein 1 (UCP1) and adipogenic transcription factors PPARgamma and CCAAT/enhancer-binding proteins (C/EBPs), and induction of mitochondrial biogenesis via p38 mitogen-activated protein (MAP) kinase-(also known as Mapk14) and PGC-1-dependent pathways. Moreover, BMP7 triggers commitment of mesenchymal progenitor cells to a brown adipocyte lineage, and implantation of these cells into nude mice results in development of adipose tissue containing mostly brown adipocytes. Bmp7 knockout embryos show a marked paucity of brown fat and an almost complete absence of UCP1. Adenoviral-mediated expression of BMP7 in mice results in a significant increase in brown, but not white, fat mass and leads to an increase in energy expenditure and a reduction in weight gain. These data reveal an important role of BMP7 in promoting brown adipocyte differentiation and thermogenesis in vivo and in vitro, and provide a potential new therapeutic approach for the treatment of obesity.

Figure 1. BMP-7 induces brown, but not white, preadipocyte differentiation and the essential role of p38 MAPK in BMP-7-induced thermogenesis

a, Oil Red O staining of brown preadipocytes and 3T3-L1 white preadipocytes grown in growth medium supplemented with BMPs or vehicle (control) for 8 days. b, Quantitative-RT-PCR (Q-RT-PCR) analysis for UCP-1 and Runx2 in brown preadipocytes treated with vehicle or BMPs in combination of insulin and T3 for 7 days. c, Q-RT-PCR analysis for PGC-1α and UCP-1 in response to 4 hrs of cAMP stimulation in brown preadipocytes differentiated in growth medium supplemented with BMP-7. Data are presented as mean ± SEM (n = 3). Asterisks depict statistically significant differences between control and experimental groups (* P < 0.05, ** P < 0.01, *** P < 0.001). d, Western blot analysis of phosphorylation of Smad1/5/8, p38 MAPK, and ATF-2 in response to 0, 10, and 30 min of BMP-7 stimulation in brown and white preadipocytes. e, Western blot analysis of UCP-1 in brown preadipocytes cultured in growth medium supplemented with vehicle or BMP-7 for 10 days. Three p38 MAPK inhibitors or vehicle (DMSO) were added to the cells 7 hrs prior to and throughout BMP-7 treatment. Cyclophilin A (Cyp A) serves as a loading control.

Figure 2. Molecular mechanisms by which BMP-7 induces brown adipogenesis and mitochondrial biogenesis

Q-RT-PCR analysis for genes served as (a) early adipogenic inhibitors, (b) adipogenic markers common to brown and white fat, (c) brown fat-specific markers, and (d) mitochondrial components in brown preadipocytes treated with vehicle (control) or BMP-7 for 3 or 8 days. Data are presented as mean ± SEM (n = 3). (* P < 0.05, ** P < 0.01, *** P < 0.001). e, Transmission electron microscopy of brown preadipocytes treated with vehicle or BMP-7 for 9 days. Original magnification = 24,000 X.

Figure 3. BMP-7 triggers commitment of mesenchymal progenitor cells to brown adipocyte lineage in vitro and in vivo

a, Oil Red O staining and Western blotting analysis for UCP-1 in C3H10T1/2 cells treated with BMP-7 or vehicle (control) for 3 days followed by adipogenic induction for 7 days. β-tubulin serves as a loading control. b, c, Q-RT-PCR analysis for genes involved in adipogenic program (b) and mitochondrial biogenesis (c) in cells described in a. Data are presented as mean ± SEM (n = 3). d, UCP-1 immunohistochemical staining on a tissue derived from implantation of BMP-7-treated C3H10T1/2 cells into nude mice. Original magnification = 400 X.

Figure 4. Evidence for an essential role of BMP-7 in BAT development and regulation of whole body energy expenditure by loss-of-function and gain-of-function approaches

a, Gross morphological analysis of BAT and liver from 1-day old wild-type and BMP-7 KO pups. b, Transverse histological sections at the thoracic region from wild-type and BMP-7 KO embryos at 17.5 dpc. Slides were stained by HΣE. Arrows indicate BAT. Original magnification is 200 X. c, Western blotting analysis of UCP-1 and insulin receptor β chain in BAT from wild-type and BMP-7 KO embryos at 18.5 dpc. β-tubulin serves as a loading control. d, Adenoviruses expressing BMP-7, BMP-3 or LacZ control were injected into 4-week old C57BL/6 mice via the tail vein (n = 5). Energy Expenditure was determined at 10 days after injection by indirect calorimetry. Basal body temperature was measured using a rectal thermometer after 14 days of injection. Percentage of body weight change at day 5 after adenoviral injection was determined by Wilcoxon Signed Rank test. e, Adenoviruses expressing BMP-7 or LacZ control were injected into 12-week old C57BL/6 mice via the tail vein (n = 6). Mice were sacrificed 15 days after injection. Expression of PRDM16 and UCP-1 in BAT was measured by Q-RT-PCR. Data are presented as mean ± SEM. Asterisks depict statistically significant differences between control and BMP-7 groups (* P < 0.05, ** P < 0.01, *** P < 0.001). f, Proposed model for the role of BMPs in determination of brown versus white adipocyte development.